The present invention relates to ongoing efforts to produce rat genetic models through gene targeting technology. More particularly, the invention relates to a transgenic line of rats that express a gene of interest, e.g., enhanced green fluorescent protein (EGFP), exclusively in the germ-line, and to genetically modified spermatogonial stem cells obtained from these transgenic rats.
Germ cells have the ability to transmit genetic information from one generation to the next. Currently, there are no known markers for germ cells that are either expressed throughout germ cell development or that are exclusive to the germ cells.
The precursors to germ cells, primordial germ cells (PGCs), have been shown to contain high alkaline phosphatase activity (AP). Other molecules, such as oct3/4, stage specific embryonic antigen 1 (SSEA1), fragillis, nanog, nanos, vasa and germ cell nuclear antigen (GCNA), also have been suggested as reliable molecular markers for identifying developing germ cells (Ref. 1-10). Yet AP, oct3/4, SSEA1, nanog, and fragillis are expressed as well in undifferentiated cells of the early mammalian embryo (blastocyst to epiblast in the mouse); hence, they do not distinguish emerging germ cells from other undifferentiated cells of the embryo. Markers such as vasa and GCNA are germ cell-specific but are not expressed during all stages of germ cell development and GCNA is sex-specific. Thus, a single molecule has not been reported in mammals that marks the germ cell lineage throughout development.
The laboratory rat represents one of the most comprehensively studied mammalian species, with described use in more than a million publications in a wide range of medically relevant areas. Qualities such as size, fecundity, behavior, ease of surgical techniques, tissue sampling and general laboratory management have contributed to its popularity (Refs. 11-13). The rat is a much better animal model than the mouse to study human physiology and disease. However, failures in culturing pluripotent ES cells from the rat has resulted in the mouse becoming a widely popular animal model, because mouse embryonic stem (ES) cells can renew with a sense of immortality. An alternative to ES cells in rat model would be spermatogonial stem cells. In the past, however, rat spermatogonia could not be propagated in culture and had to be isolated fresh from rats for experimentation.